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A CRISPR/Cas12a-based colorimetric AuNPs biosensor for naked-eye detection of pathogenic bacteria in clinical samples
Laibao Zheng1, Chaochuan Zheng2, Weiwei Wang1
1Wenzhou Key Laboratory of Sanitary Microbiology, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, China.
Abstract:
Pathogenic bacteria, such as Pseudomonas aeruginosa, pose significant threats to public health due to their multidrug resistance and association with severe infections. Rapid and reliable detection methods are crucial for timely treatment and effective infection control, especially in resource-limited settings. In this study, we developed a CRISPR/Cas12a-based colorimetric biosensor that leverages Cas12a's trans-cleavage activity to release left single-stranded DNA (lDNA). The released lDNA facilitates hybridization with clDNA-functionalized gold nanoparticles (AuNPs), resulting in a visible color change. The biosensor achieved a detection limit of 100 CFU/reaction for P. aeruginosa within 2 hours, with excellent specificity and robustness, as validated in spiked sputum and blood samples. Clinical testing using 32 blood samples (13 positive, 19 negative) confirmed its high diagnostic accuracy, achieving an AUC of 1 in ROC curve analysis. The platform's simplicity, robustness, and programmability suggest its broad potential for rapid infectious disease diagnostics, particularly in low-resource settings.
Insights
A new CRISPR/Cas12a biosensor detects Pseudomonas aeruginosa in two hours. This rapid, colorimetric method shows high accuracy in clinical samples, offering a promising tool for infectious disease diagnostics.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Pseudomonas aeruginosa is a multidrug-resistant pathogen causing severe infections.
- Rapid detection is vital for treatment and infection control, especially in resource-limited areas.
- Existing methods can be slow or require specialized equipment.
Purpose of the Study:
- To develop a rapid, sensitive, and specific CRISPR/Cas12a-based colorimetric biosensor for P. aeruginosa detection.
- To evaluate the biosensor's performance in spiked clinical samples (sputum, blood).
- To assess the diagnostic accuracy of the biosensor in a clinical setting.
Main Methods:
- Utilized CRISPR/Cas12a system leveraging trans-cleavage activity to release single-stranded DNA (ssDNA).
- Developed a colorimetric assay involving hybridization of released ssDNA with functionalized gold nanoparticles (AuNPs).
- Validated specificity and robustness using spiked sputum and blood samples, and clinical blood samples.
Main Results:
- Achieved a detection limit of 10^0 CFU/reaction for P. aeruginosa within 2 hours.
- Demonstrated excellent specificity and robustness in spiked samples.
- Clinical testing on 32 blood samples showed high diagnostic accuracy with an AUC of 1.
Conclusions:
- The developed CRISPR/Cas12a biosensor provides rapid and accurate detection of P. aeruginosa.
- The platform's simplicity and robustness make it suitable for resource-limited settings.
- This technology holds significant potential for point-of-care infectious disease diagnostics.
